Sensitivity enhancement of CUPRAC and iron(III)-phenanthroline antioxidant assays by preconcentration of colored reaction products on a weakly acidic cation exchanger

The chemical diversity of antioxidants (acting as non-enzymatic defenses of an organism to oxidative stress) makes it difficult to separate and quantify antioxidants from the vegetable matrix. We report the development of simple and widely applicable total antioxidant capacity (TAC) assays of increased sensitivity quantitating dietary polyphenols, vitamins C and E from dilute solutions utilizing the copper(II)-neocuproine (Cu(II)-Nc) and iron(III)-phenanthroline (Fe(III)-phen) reagents as chromogenic oxidants. The sensitivities of the CUPRAC (cupric ion reducing antioxidant capacity) method for TAC, and of Fe(III)-phen method for ascorbic acid assays were enhanced by preconcentration of colored species derived from the redox reaction of chromogenic oxidant with the tested antioxidant. The proposed method was useful in concentrating the colored cations derived from the antioxidant values of dilute solutions where ordinary TAC assays gave negligible absorbances. When an antioxidant reduced the chromogenic reagents Cu(II)-Nc or Fe(III)-phen, the resulting large cations of Cu (Nc)₂ ⁺and Fe (phen)₃ ^{2 +}were selectively retained by a weakly acidic cation exchanger Amberlite IRC-50 resin in Na⁺-form. The ferrous-phenanthroline and cuprous-neocuproine cations were retained at pH 6 and 7, respectively, on IRC-50 resin. The resin-retained species were eluted with (1:1 (v/v) EtOH - 1 M aqueous HCl) and (1:1 (v/v) EtOH + 1 M aqueous HClO₄) solutions, respectively, for ferrous and cuprous species, yielding preconcentration factors between 5 and 10. The Fe (phen)₃ ^{2 +}-containing eluate was adjusted to pH 6 and its absorbance measured at 510 nm, while the absorbance of the Cu (Nc)₂ ⁺-containing eluate was directly measured at 450 nm. The proposed assays were rapid, and the formed complexes were stable. Preconcentration of antioxidant solutions by the proposed 'sorption-spectrophotometry' increased analytical sensitivity.